An essential step in eukaryotic gene expression is pre-mRNA splicing, which involves the removal of introns and ligation of exons. Mutations that cause errors in splicing are found in at least 15% of human genetic diseases; additional diseases are caused by mutations in trans-acting splicing factors. Members of the DExD/H family of nucleic acid-dependent ATPases play essential roles in splicing; other members of this protein family participate in all aspects of cellular activities involving nucleic acids, including DNA replication, recombination, transcription, mRNA transport, translation, and RNA processing. Residues in DExD/H box proteins that are essential for function have been characterized; however, much less is known about how the proteins act in vivo.The goal of this AREA proposal is to identify and characterize co-factors that modulate the activity of one member of this protein family, Prp43. Prp43 is encoded by an essential gene in yeast and has homologues in other eukaryotes, including humans. Although highly conserved with other spliceosomal ATPases, Prp43 may participate in cellular pathways in addition to splicing. Its target(s) and how it achieves specificity within the spliceosome and other complexes in which it has been identified is not known. Clearly, to fully understand the activity and function of Prp43 and other DExD/H proteins, the cofactors that interact with them in vivo must be identified and characterized. We plan to use biochemical and genetic approaches to: 1) Identify the spliceosome interaction domain(s) of Prp43 2) Identify and characterize potential cofactors for Prp43 Understanding the target of Prp43 RNA-dependent ATPase activity within the spliceosome and other cellular complexes will assist in understanding how the human homologue of Prp43, as well as related DExD/H-box proteins, achieve target specificity in vivo.